The clotting of blood is part of the body""s natural response to injury or trauma. Blood clot formation derives from a series of events called the coagulation cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure which forms the insoluble framework of the blood clot. As a part of hemostasis, clot formation is often a life-saving process in response to trauma and serves to arrest the flow of blood from severed vasculature.
The life-saving process of clot production in response to an injury can become life-threatening when it occurs at inappropriate places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blood to an organ or other body part. In addition, the deposition of fibrinogen contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood flow. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, pathologies of blood coagulation, such as heart attacks, myocardial infarctions, strokes, and the like, have been estimated to account for approximately fifty percent of all hospital deaths.
Fibrinogen is synthesized and secreted into the circulation by the liver. Circulating fibrinogen is polymerized under attack by thrombin to form fibrin, which is the major component of blood clots or thrombi. Subsequently, fibrin is depolymerized under attack by plasmin to restore the fluidity of the plasma. Many of the steps in the polymerization and depolymerization processes have been well established, Doolittle et al., Annu. Rev. Biochem., 53:195-229 (1984). The elevated levels of fibrinogen which are part of the acute phase response occurring in the wake of infections and trauma are now known to come from increased hepatic production, primarily in response to interleukin-6 (IL-6). Seghal et al., Ann N.Y. Acad. Sci. 557:1-583.
Fibrinogen, one of the more well-studied proteins, plays a central role in clot formation and wound healing. It has a complex structure which includes a heavily disulfide-bonded hexamer composed of two copies each of the xcex1, xcex2 and xcex3 subunits. Recently, new attention has been given to structure/function relationships in the fibrinogen molecule. This new interest has in part been prompted by growth in the understanding of this protein""s range of activity in normal and pathological states, see for example, Blomback et al., Biotechnology of Blood, 225-279 (1991), Bini et al., Ann N.Y Acad. Sci., 667:112-126 (1992) and Dvorak et al., Ann N.Y. Acad. Sci., 667:101-111 (1992).
By the late 1960""s, the general subunit structure of fibrinogen was firmly established. Blomback et al., Nature 218:130-134. A decade later, the complete amino acid sequence was reported. Lottspeich et al., Hoppe-Seyler""s, Physiol. Chem. 358:935-938 (1997), Henschen et al., Hoppe-Seyler""s, Physiol Chem., 358:1643-1646, Henschen et al., Hoppe-Seyler""s, Physiol Chem., 360:1951-1956, Doolittle et al., Nature, 280:464-468 (1979). Over the next 10 years, the cluster of three separate genes encoding the xcex1 (alpha), xcex2 (beta) and xcex3 (gamma) subunits was identified on chromosome 4q23-q32, Kant et al., Proc. Natl. Acad. Sci. USA, 82:2344-2348 (1985), and the apparently complete genetic sequences of all three fibrinogen subunits were published. Chung et al., Adv. Exp. Med. Biol., 281:39-48 (1991). These studies indicated that the a subunit lacked a globular C-terminal domain comparable to those present in the xcex2 and xcex3 subunits.
The subsequent discovery of an additional exon (i.e., exon VI) downstream from the established a subunit gene has resolved the evolutionary mystery posed by the imperfectly parallel structure of the three major subunits. Fu et al., Biochemistry, 31:11968-11972 (1992), Weissbach et al., Proc. Natl. Acad. Sci. USA, 87:5198-5202 (1990). A novel fibrinogen xcex1 chain transcript has been identified at low frequency bearing the exon VI-derived sequences as a separate open reading frame. Additional splicing leads to the use of this extra sequence to elongate the xcex1 chain by 35% (236 similar to those of the xcex2 and xcex3 chains.
A major impetus to fibrinogen research has been provided by the recent identification of this long overlooked, naturally occurring elongated version of the xcex1 subunit, designated xe2x80x9cxcex1Exe2x80x9d. See Fu et al., Biochemistry, 31:11968-11972 (1992). Evidence shows that the xcex1E chain is assembled into fibrinogen molecules and that its synthesis is enhanced by interleukin-6 (IL-6). These facts suggest that the xcex1E subunit participates in both the acute phase response and in normal physiology.
Using a polyclonal rabbit antibody preparation specific to the VI-domain or xcex1EC domain, xcex1E was demonstrated to occur in plasma fibrinogen as part of (xcex1Excex2xcex3)2, a homodimeric (i.e., symmetrical) molecule of 420 kilodaltons (kDa). Fu et al., Proc. Natl. Acad. Sci. USA, 91:2625-2628 (1994). This species has been designated xe2x80x9cfibrinogen-420xe2x80x9d (xcex1Excex2xcex3)2 to distinguish it from the abundant 340 kDa form of fibrinogen, denoted xe2x80x9cfibrinogen-340xe2x80x9d(xcex1xcex2xcex3)2). Fibrinogen-420 accounts for approximately 1% of the total fibrinogen in normal adult plasma and 3% of the total in umbilical cord plasma. Grieninger et al., Blood, 90:2609 (1997). The relatively low circulating level of fibrinogen-420 is undoubtedly responsible for its having escaped detection. These two xcex1EC domains that distinguish Fibrinogen-420 from Fibrinogen-340 are likely to significantly influence the fibrinogen molecule""s multiple binding capacities and functions.
Transcripts encoding fibrinogen subunit counterparts having exceptionally high C-terminal homology to human xcex1E have been detected thus far in lamprey, where it arises from a second xcex1 gene, as well as in chicken, rabbit, rat, and baboon. See Pan et al., Proc. Natl. Acad. Sci. USA, 89:2066-2070 (1992), Doolittle et al., Thromb. Res., 68:489-493 (1992) and Fu et al., Genomics 30:71-76 (1995). This degree of xcex1 subunit-associated globular domain preservation in the vertebrate genome signals an important, if as yet unknown, role for xcex1E. Clues to its potential significance may lie in the similarity of the extension in xcex1E, not only to the corresponding regions of the fibrinogen and chains, but also to carboxy domains of a number of non-fibrinogen proteins from fruit fly to man. Chung et al., Biochemistry, 22:3244-3250 (1983), Chung et al., Biochemistry 22:3250-3256 (1983), Baker et al., Science 250:1370-1377 (1990), Koyama et al., Proc. Natl. Acad. Sci. USA, 84:1609-1613 (1987), Morel et al., Proc. Natl. Acad. Sci. USA, 86:6582-6586 (1989), Nies et al., J. Biol. Chem., 266:2818-2823 (1991), Norenberg et al., Neuron, 8:849-863 (1992), Xu et al., Proc. Natl. Acad. Sci. USA, 87:2097-2101. Where functions are known, these non-fibrinogen proteins are constituents of the extracellular matrix and have adhesive properties. It is expected that continued research will permit the determination of whether the xcex1E globular domain contributes in a subtle way to the primary function of fibrinogen (clot formation and wound healing) or, following the example of other differentially used exons, promotes an alternative function. Chan et al., Science, 254:1382-1385 (1991), Descombes et al., Cell, 67:569-579 (1991), Early et al., Cell, 20:313-319 (1980). Thus there is a need to isolate fragments of the Fibrinogen-420 molecule.
In clinical settings it is commonly desirable to activate or potentiate the fibrinolytic system. This is particularly necessary in cases of myocardial infarction in which coronary arteries become occluded and require recanalization. Catheterization has proven somewhat effective in such recanalization, but pharmacologic agents are desired to supplement or replace such invasive procedures to inhibit reocclusion. The study of the intricate system of thrombolysis and fibrinolysis has been a rapidly growing field, which has resulted in the development of a new generation of thrombolytic agents.
Previous therapeutic treatments for dissolving life-threatening clots have included injecting into the blood system various enzymes which are known to break down fibrin. Collen D, Circulation, 93:857-865 (1996). The problems with these treatments has been that the enzymes were not site-specific, and, therefore, would do more than just cause dissolution of the clot. In addition, these enzymes interfere with and destroy many vital protein interactions that serve to keep the body from bleeding excessively due to the many minor injuries it receives on a daily basis. Destruction of these safeguards by such enzymes can lead to serious hemorrhage and other potentially fatal complications.
Currently, the best known therapeutic agents for inducing or enhancing thrombolysis are compounds which cause the activation of plasminogen, the so-called xe2x80x9cplasminogen activators,xe2x80x9d Brakman et al., Ann NY Acad Sci, vol. 667 (1992). These compounds cause the hydrolysis of the arg560-val561 peptide bond in plasminogen. This hydrolysis yields the active two-chain serine protease, plasmin. Both plasmin and plasminogen activator are produced endogenously in a mammal. A number of such plasminogen activators are known, including serine proteases such as urokinase plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), streptokinase (a non-enzyme protein) and staphylokinase. Of these, streptokinase is the most widely used therapeutic thrombolytic agent. However, while streptokinase and the other plasminogen activators have proven helpful in recanalization of coronary arteries, their ability to improve mortality is not devoid of side effects and their use still requires stringent control conditions to achieve success in a high percentage of cases, Martin et al., Chapter 72 in Hemostasis and Thrombisis: Basic Principles and Clinical Practice, 3rd ed., (1994). In addition, the use of such compounds can cause bleeding complications in susceptible individuals.
Elevated levels of fibrinogen have been found in patients suffering from clinically overt coronary heart disease, stroke and peripheral vascular disease. Although the underlying mechanisms remain speculative, recent epidemiological studies leave little doubt that plasma fibrinogen levels are an independent cardiovascular risk factor possessing predictive power which is at least as high as that of other accepted risk factors such as smoking, hypertension, hyperlipoproteinemia or diabetes. Ernst et al., J. Internal Med., 227:365-372 (1990), Ernst et al., Ann Intern. Med. 118:956-963 (1993).
During myocardial infarctions (M.I.) certain blood iso-enzymes including CK (Creatine Phosphokinase)-MB(Muscle-Brain) are used to confirm the diagnosis of a M.I. in a subject as well as other parameters like electrocardiograms (ECG). These enzymes are often elevated, and must be monitored carefully for more than 72 hours. Treatment will be continued for this time or longer until a definitive diagnosis can be made. Thus, new methods are needed to accurately confirm suspected myocardial infarctions.
The structure of fibrin has been analyzed extensively in vitro by Doolittle et al., Annu Rev Biochem 53:195-229 (1984). Only recently, however, has attention been paid to the molecular structure of human thrombi and atherosclerotic plaques with respect to fibrinogen and fibrin products, Bini et al., Blood 69:1038-1045 (1987). Whereas thrombi formed in vivo consist primarily of fibrin II cross-linked by factor XIIIa, fibrinogen itself is a major component of uncomplicated atherosclerotic lesions, particularly fibrous and fatty plaques. Immunohistochemical as well as immunoelectrophoretic analyses indicate that fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue transglutaminase without becoming converted to fibrin, Valenzuela et al., Am. J Pathol. 141:861-880 (1992). Further understanding of these issues awaits the development of methods for the differential determination of fibrinogen subtypes in medical samples.
In wound repair, fibrinogen serves as a key protein, achieving rapid arrest of bleeding following vessel injury. It promotes both the aggregation of activated platelets with one another to form a hemostatic plug, as well as endothelial cell binding at the site of injury to seal the margins of the wound. As the most abundant adhesive protein in the blood, fibrinogen attaches specifically to platelets, endothelial cells and neutrophils via different integrins, Hynes et al., Cell, 69:11-25 (1992). Five putative receptor recognition domains on human fibrinogen, distributed over its three subunits, have been identified by in vitro and in vivo analyses. Kloczewiak et al., Biochemistry 23:1767-1774 (1984), Cheresh et al., Cell, 58:945-953 (1989), Loike et al., Proc Natl Acad Sci USA, 88:1044-1048 (1991), Farrell et al., Proc Natl Acad Sci USA, 89:10729-10732 (1992), Gonda et al., Proc Natl Acad Sci USA, 79:4565-4569 (1982), Ribes et al., J. Clin Invest., 84:435-442 (1989). In fibrinogen which contains the variant xcex1E chains, masking of these sites, as well as addition of new sites, are distinct possibilities with ramifications that must be explored.
As a result of the foregoing, there exists a need for a better understanding of the structure and function of fibrinogen, especially in relation to the fibrinogen-420xcex1E C domain. There also exists a need for isolating and purifying fragments of fibrinogen-420xcex1E C. Diagnosis and treatment of disease states associated with physiological processes involving fibrinogen-420 and fibrin-420 are lacking. The present invention effectively addresses these and other needs for the first time.
This invention relates to a diagnostic method, for characterizing fibrinogen, the method includes analyzing a sample, such as biological fluids and tissue, for xcex1ECX fragments. Such analysis typically includes contacting the sample with at least one monospecific antibody that binds to an xcex1E C domain where specific binding of the antibody indicates the presence of the xcex1ECX cleavage fragments of fibrinogen. These xcex1ECX cleavage fragments of fibrinogen are defined by the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2 in the sample. Presence of xcex1ECX cleavage fragments indicates proteolytic degradation of fibrinogen-420, in vivo or in vitro and can be used to diagnose a myocardial infarction in a mammal. The diagnostic method of the present invention can also be used to regulate the amount of plasminogen activator or plasmin given to a mammal in vivo. In one preferred embodiment, the monospecific antibody that binds to an xcex1EC domain of fibrinogen can further be detectably labeled with a detectable marker moiety. In another exemplary embodiment, the proteolytic enzyme includes plasminogen, plasminogen activator, fibrinolytic metalloproteinases, u-PA, t-PA, r-PA, n-PA, streptokinase, staphylokinase and combinations thereof.
The present invention also provides fibrinogen cleavage fragments, Fibrinogen-420xcex1ECX fragments. These xcex1ECX cleavage fragments of fibrinogen include amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2.
The invention also provides xcex1ECX cleavage fragments of amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2, conjugated to a carrier for administration to a subject.
In one embodiment of the invention, xcex1ECX cleavage fragments of amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2, are admixed with a physiologically acceptable diluent.
The invention further relates to a method of purifying xcex1ECX fragments of fibrinogen-420 which includes contacting fibrinogen with a proteolytic enzyme to provide fragments of the fibrinogen, and selectively removing the xcex1ECX cleavage fragments of fibrinogen defined by the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2 from the sample.
In one preferred embodiment, the proteolytic enzyme can be fibrinolytic matrix metalloproteinase, plasmin, plasminogen activator u-PA, t-PA, r-PA, n-PA, streptokinase, staphylokinase an combinations thereof. The method of the present invention can also be performed in vitro or in vivo.
The invention further relates to a method of purifying fibrinogen which includes contacting fibrinogen with a proteolytic enzyme to provide fragments of the fibrinogen, contacting the fibrinogen fragments with at least one monospecific antibody that binds to an xcex1EC domain of fibrinogen where specific binding of the antibody indicates the presence of the xcex1ECX cleavage fragments of fibrinogen-420 and selectively removing the xcex1ECX cleavage fragments of fibrinogen defined by the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2 from the sample.
The present invention can also be used to detect xcex1ECX fragments in vivo or in vitro. This method includes contacting fibrinogen with plasmin or a plasminogen activator to provide fragments of the fibrinogen, then contacting the fragments of fibrinogen with at least one monospecific antibody that binds to an xcex1EC domain of fibrinogen, where specific binding of the antibody indicates the presence of the xcex1ECX cleavage fragments of fibrinogen defined by the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or homologs having at least 90% identity with SEQ ID NO: 2 in the sample.
In one preferred embodiment, the monospecific antibody that binds to an xcex1EC domain of fibrinogen can further be detectably labeled with a detectable marker moiety. In another preferred embodiment, the present invention can also be used to detect xcex1ECX fragments in vivo, for example, in a mammal suffering from a myocardial infarction. The presence of xcex1ECX cleavage fragments in a sample of blood indicates that fibrin(ogen)olysis has occurred.
In yet another embodiment of the invention, a monospecific antibody is provided which binds with an epitope of the xcex1ECX cleavage fragment of fibrinogen. This monospecific antibody can be monoclonal. Preferably such antibodies can be labeled with a detectable moiety such as radioactive labels, enzymes, specific binding pair components, colloidal dye substances, fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, chromophores, affinity columns and the like.
In another embodiment of the invention, a nucleic acid comprising nucleotide SEQ ID NO: 5, is provided. This nucleic acid encodes xcex1ECX cleavage fragments set forth in: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or homologs having at least about 90% identity with SEQ ID NO: 2. The nucleic acid can be isolated, natural or synthetic DNA or RNA encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a homolog having at least about 90% identity with SEQ ID NO:2.
The invention also includes a vector for transfecting a host cell to express heterologous or recombinant proteins including the DNA segment encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or homologs having at least about 90% identity with SEQ ID NO: 2, which is conjugated to a promoter.
The method of the present invention includes a method of making a host cell which expresses a heterologous or recombinant protein which includes transfecting the cell with a vector including a DNA segment encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or homologs having at least about 90% identity with SEQ ID NO:2, conjugated to the promoter.
Still another embodiment of the present invention is a method for treating a mammal suffering from conditions or pathologies related to fibrinogen metabolism by administering an effective amount of a composition which includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or homologs having at least 90% identity with SEQ ID NO:2.
These and other advantages of the present invention will be appreciated from the detailed description and examples which are set forth herein. The detailed description and examples enhance the understanding of the invention, but are not intended to limit the scope of the invention.